Development and application of an efficient recombineering system for Burkholderia glumae and Burkholderia plantarii

Abstract

The lambda phage Red proteins Redα/Redβ/Redγ and Rac prophage RecE/RecT proteins are powerful tools for precise and efficient genetic manipulation but have been limited to only a few prokaryotes. Here, we report the development and application of a new recombineering system for Burkholderia glumae and Burkholderia plantarii based on three Rac bacteriophage RecET-like operons, RecEThe_BDU8, RecETh_TJI49 and RecETh1h2e_YI23, which were obtained from three different Burkholderia species. Recombineering experiments indicated that RecETh_TJI49 and RecETh1h2e_YI23 showed higher recombination efficiency compared to RecEThe_BDU8 in Burkholderia glumae PG1. Furthermore, all of the proteins currently categorized as hypothetical proteins in RecETh1h2e_YI23, RecETh_TJI49 and RecEThe_BDU8 may have a positive effect on recombination in B. glumae PG1 except for the h2 protein in RecETh1h2e_YI23. Additionally, RecET_YI23 combined with exonuclease inhibitors Pluγ or Redγ exhibited equivalent recombination efficiency compared to Redγβα in Escherichia coli, providing potential opportunity of recombineering in other Gram-negative bacteria for its loose host specificity. Using recombinase-assisted in situ insertion of promoters, we successfully activated three cryptic non-ribosomal peptide synthetase biosynthetic gene clusters in Burkholderia strains, resulting in the generation of a series of lipopeptides that were further purified and characterized. Compound 7 exhibited significant potential anti-inflammatory activity by inhibiting lipopolysaccharide-stimulated nitric oxide production in RAW 264.7 macrophages. This recombineering system may greatly enhance functional genome research and the mining of novel natural products in the other species of the genus Burkholderia after optimization of a protocol.